Methods of inducing differentiation of stem cells into a specific cell lineage

ABSTRACT

The present invention relates to methods of inducing differentiation of stem cells into a specific cell lineage, preferably lung cells. In particular, the invention relates to in vitro methods of inducing differentiation of stem cells into a specific cell lineage. The invention also relates to methods of producing and recovering differentiated stem cells of a specific cell lineage. The invention also includes differentiated stem cells and cell lineages produced by the methods of the present invention. In one aspect of the present invention there is provided a method of inducing differentiation of a stem cell into a specific cell lineage, preferably lung cells, the method including: culturing a stem cell in vitro in the presence of a tissue sample and/or extracellular medium of a tissue sample, under conditions that induce differentiation of the stem cell into a specific cell lineage, wherein the differentiated stem cell is the same cell type as the tissue sample.

The present invention relates to methods of inducing differentiation ofstem cells into a specific cell lineage. In particular, the inventionrelates to in vitro methods of inducing differentiation of stem cellsinto a specific cell lineage. The invention also relates to methods ofproducing and recovering differentiated stem cells of a specific celllineage. The invention also includes differentiated stem cells and celllineages produced by the methods of the present invention.

INTRODUCTION

Stem cells are undifferentiated cells which can give rise to asuccession of mature functional cells. Embryonic stem (ES) cells arederived from the embryo and in the mouse, when maintained in vitro inthe presence of leukocyte inhibitory factor (LIF) are pluripotent, thuspossessing the capability of developing into any organ, cell type ortissue type. Furthermore, when grown in hanging drops as a cellaggregate in the absence of LIF, mouse ES cells differentiate intorepresentatives of all three embryonic germ layers, namely endoderm,mesoderm and ectoderm. Such aggregates are thus called embryoid bodies(EBs).

The process of differentiation in stem cells involves selectivedevelopment of immature cells to committed and fully mature cells ofvarious cell lineages. Derivatives of such cell lineages include,respiratory, muscle, neural, skeletal, blood (hematopoietic),endothelial and epithelial cells. Differentiation of stem cells is knownto be triggered by various growth factors and regulatory molecules.During differentiation the expression of stem cell specific genes andmarkers are often lost and cells acquire gene expression profiles ofsomatic cells or their precursors. In some cases, “master” genes havebeen described which control differentiation versus self-renewal.

Whilst differentiation of stem cells into various cell lineages may beinduced with a degree of certainty, a differentiation outcome of apopulation of stem cells is less predictable. Placing the cells underconditions which induce specific cell types has been one form of anattempt to regulate the differentiation outcome. These conditionstypically include growing the cells to high or low density, changingmedia, introducing or removing cytokines, hormones and growth factors,creating an environment which suits differentiation toward a specificcell type, such as providing a suitable substrate.

Generally, when a stem cell culture is induced to differentiate, thedifferentiated population is analyzed for particular cell types byexpression of genes, markers or phenotypic analysis. The respective celltypes are then typically selectively cultured to enrich their percentagepopulation to eventually obtain a pure cell type and culture. However,recovering differentiated cells of a specific cell lineage in thismanner is time-consuming and complicated.

The recovery of differentiated stem cells of a specific cell lineage canbe useful for transplantation or drug screening and drug discovery invitro and in vivo. Methods of inducing differentiation of stem cells anddifferentiated cells produced therefrom may be used for the study ofcellular and molecular biology of tissue development, for the discoveryof genes, proteins, such as differentiation factors that play a role intissue development and regeneration.

In particular, the induction of stem cells to differentiate into aspecific cell lineage is useful for transplantation and therapeuticpurposes, as well as providing potential human disease models in culture(e.g. for testing pharmaceuticals). The induction of differentiation ofstem cells into a specific cell lineage is especially useful indeveloping therapeutic methods and products for tissue specific diseasesand conditions.

Therefore there remains a need for providing effective methods ofinducing differentiation of stem cells in vitro into a specific celllineage, and then preferably providing efficient and reliable methods ofrecovering differentiated stem cells of a specific cell lineage.

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided a method ofinducing differentiation of a stem cell into a specific cell lineage,the method including:

-   -   culturing a stem cell in vitro in the presence of a tissue        sample and/or extracellular medium of a tissue sample said        sample selected during organogenesis of the tissue, under        conditions that induce differentiation of the stem cell into a        specific cell lineage.

Preferably, the tissue sample is treated to form tissue cells in asubstantially single cell suspension. Alternatively, the tissue sampleis prepared as a sheet prior to culturing with the stem cells. Thetissue cells are preferably derived from embryonic, foetal orpost-partum tissue. Most preferably, the tissue cells are mesenchymalcells. The tissue cells are preferably derived from embryonic lungmesenchyme.

The stem cells used in the methods of the present invention arepreferably embryonic stem (ES) cells. The tissue cells and/or the stemcells used in the methods of the present invention may be tagged.Preferably, the stem cells used express a transgenic marker protein thatallows for identification of differentiated stem cells. The stem cellsmay be induced to differentiate into specific cell lineages, preferablyselected from the group consisting of respiratory, pancreatic, mammary,renal, intestinal, and hepatic.

In another aspect of the present invention there is provided a method ofinducing differentiation of a stem cell into a specific cell lineage,the method including the steps of:

-   -   mixing a first sample of stem cells with a second sample of        tissue cells said cells selected during organogenesis of the        tissue to form a cell mixture;    -   culturing the cell mixture in vitro, under conditions that        induce differentiation of a stem cell into a specific cell        lineage.

Preferably, the tissue cells are in a substantially single cellsuspension prior to mixing with the stem cells. Alternatively, thetissue cells are prepared as a sheet for wrapping an undifferentiatedembryoid body. Undifferentiated embryoid bodies are preferably preparedby cultivating ES cells in hanging drops in the presence of LIF.Preferably, the culturing step includes allowing the cell mixture togrow on a permeable membrane, wherein the membrane is in contact with aculture medium, such that the stem cells are induced to differentiateinto a specific cell lineage.

In another aspect of the present invention there is provided a method ofproducing differentiated stem cells of a specific cell lineage, themethod including:

-   -   culturing stem cells in vitro in the presence of a tissue sample        and/or extracellular medium of a tissue sample, said        organogenesis        stem cell into a specific cell lineage; and    -   recovering differentiated stem cells of a specific cell lineage.

Preferably, the tissue sample is treated to form tissue cells in asubstantially single cell suspension prior to culturing with the stemcell. Alternatively, the tissue cells are prepared as a sheet forwrapping an undifferentiated embryoid body.

In a preferred aspect of the present invention there is provided amethod of producing differentiated stem cells of a specific celllineage, the method including:

-   -   culturing stem cells in vitro in the presence of tissue cells,        said cells selected during organogenesis of the tissue, under        conditions that induce differentiation of a stem cell into a        specific cell lineage; and    -   recovering the differentiated stem cells of a specific cell        lineage.

Preferably, the tissue cells are in a substantially single cellsuspension prior to culturing with the stem cells. Alternatively, thetissue cells are prepared as a sheet for wrapping an undifferentiatedembryoid body.

The culturing step preferably includes allowing the stem cells to growon a first surface of a permeable membrane and allowing the tissue cellsto grow on an opposite second surface of the permeable membrane, whereinthe membrane is in contact with a culture medium, such that the stemcells are induced to differentiate into a specific cell lineage.Differentiated stem cells of a specific cell lineage may then berecovered from the first surface of the permeable membrane.

In the methods of the present invention, the tissue cells may be derivedfrom embryonic, foetal or post-partum tissue. Preferably, the tissuecells are embryonic mesenchymal cells. More preferably, the tissue cellsare derived from lung mesenchyme tissue, more preferably embryonic lungmesenchyme. The stem cells used in the methods of the present inventionare preferably embryonic stem (ES) cells. The tissue cells and/or thestem cells used in the methods of the present invention may be tagged.Preferably, the stem cells used express a transgenic marker protein thatallows for identification of differentiated stem cells. The stem cellsmay be induced to differentiate into specific cell lineages, preferablyselected from the group consisting of respiratory, pancreatic, mammary,renal, intestinal, and hepatic.

The types of cell lineages will be determined by the tissues that areco-cultured with the stem cells or embryonic stem cells. The tissues andcell lineages are from tissues that can undergo organogenesis such as,but not limited to, respiratory, pancreatic, mammary, renal, intestinalor hepatic tissues.

In the methods of the present invention, the culturing step maypreferably include the addition of a growth factor to enhance stem celldifferentiation. Suitable growth factors may be preferably selected fromepidermal growth factor (EGF), hepatocyte growth factor (HGF) andfibroblast growth factors (FGFs) or steroid hormones (for example,glucocorticoids, vitamin A, thyroid hormone, androgens and estrogens).

In yet another aspect of the invention, there is provided differentiatedstem cells of a specific cell lineage produced according to the methodsas hereinbefore described. Preferably, the differentiated stem cell is alung, kidney, cardiomyocyte, mammary cell, salivary cell, , hepaticcell, intestinal cell or pancreatic cells. The present invention alsoprovides differentiated stem cells produced according to the methods ofthe invention that may be used for tissue repair, transplantation, celltherapy or gene therapy.

The present invention further provides a cell composition including adifferentiated stem cell produced by the methods of the presentinvention, and a carrier.

FIGURES

FIG. 1 Mouse ES cell/respiratory tissue aggregates. Double b-galactosidestaining (blue stain) and surfactant C immunohistochemistry (brownstain) demonstrates that the ES cell derivatives are induced to formbronchiolar duct-like structures that are immunoreactive to therespiratory specific marker (A-D). Note that after 6 days in culture (Aand B), surfactant C immunoreactivity can be observed throughout theentire bronchiolar-like duct throughout the ES cell derivativecytoplasm. After twelve days in culture, surfactant C immunoreactivityis restricted to a sub-set of the bronchiolar-like duct population andwithin these ducts, to the cell surface of the ES cell derivatives (seered arrows in C and D).

FIG. 2 Human embryonic stem (hES) cell directed surfactant C (Sp-C)expression. hEScell/mouse lung aggregates were grown for 6 days invitro. Cell nuclei are identified by the generic nuclear stain Hoechst33342 (blue), hES cell derivatives are identified by green fluorescence,and Sp-C localisation by an anti- mouse and human Sp-C specific antibody(red). Sp-C localisation is observed within the mouse tissue and withinhES derivatives.

FIG. 3 shows fifty neurospheres following plating in 35 mm dishes. (A)control media and (B) HGF+ containing media (HGF+ media is DMEMcontaining 3% charcoal stripped foetal calf serum, 10 μg/ml insulin, 1μg/ml cholera toxin, 25 ng/ml epidermal growth factor, 10 ng/mlhepatocyte growth factor and 25 ng/ml FGF7)—note foci. (C) DoubleHoechst (blue) and anti-surfactant C (red) fluorescence to revealrespiratory differentiation. (D) High power (hp) magnification of (C) tohighlight double labelling of single cells.

FIG. 4 shows reverse transcriptase—polymerase chain reaction (RT-PCR)for endodermal and respiratory markers of mouse embryoid bodies andneurospheres cultured for 8 days in DMEM+10% FCS and each of theindicated growth factor supplements.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention there is provided a method ofinducing differentiation of a stem cell into a specific cell lineage,the method including:

-   -   culturing a stem cell in vitro In the presence of a tissue        sample and/or extracellular medium of a tissue sample said        sample selected during organogenesis of the tissue, under        conditions that induce differentiation of the stem cell into a        specific cell lineage.

Applicants have found that culturing stem cells in the presence of atissue sample of a specific cell type provides an effective means ofproducing differentiated stem cells reminiscent of a specific celllineage. They have further found that the tissue is of the type that canundergo organogenesis. It is at this time that the tissue is best usedto induce differentiation of stem cells. In the methods of the presentinvention the differentiation outcome of a stem cell can be determined,as the differentiated stem cells are the same cell type (ie preferablyexpress a similar set of markers) as the tissue sample used inco-culture with the stem cells.

In the methods of the present invention the tissue sample is preferablytreated to form tissue cells in a substantially single cell suspensionprior to culturing with the stem cell. Tissue cells in a substantiallysingle cell suspension enhance the exposure and contact of secretedproducts and chemical cues produced by the tissue cells to act on andinduce differentiation of a stem cell in co-culture. The applicants havefound that tissue cells in single cell suspension that are co-culturedwith stem cells tend to form heterotypic tissue that comprisedifferentiated stem cells aggregated with the tissue cells, wherein thedifferentiated stem cells are the same cell type as the tissue cells.Furthermore, when tissue cells are prepared as a sheet in which anundifferentiated embryoid body is wrapped, the applicants have foundthat the stem cells will form a heterotypic tissue comprised of cellscharacteristic of the tissue from which the tissue sheet was derived.

The phrase “inducing differentiation of a stem cell into a specific celllineage” as used herein is taken to mean causing a stem cell to developinto a specific differentiated cell lineage as a result of a direct orintentional influence on the stem cell. Influencing factors that mayinduce differentiation in a stem cell can include cellular parameterssuch as ion influx, a pH change and/or extracellular factors, such assecreted proteins, such as but not limited to growth factors andcytokines that regulate and trigger differentiation. It may includeculturing the cell to confluence and may be influenced by cell density.

In a preferred embodiment of the invention differentiation of a stemcell into a specific cell lineage is achieved by co-culturing tissuecells in a substantially single cell suspension with stem cells topreferably form heterotypic tissue (ie differentiated stem cellsaggregated with tissue cells). Heterotypic recombinations ofdifferentiated stem cells aggregated with the tissue cells arepreferably formed, wherein the differentiated stem cells are the samecell type as the tissue cells. Tissue cells that are in a substantiallysingle cell suspension allow for enhanced induction of stem cells todifferentiate and to form heterotypic re-association in vitro with thetissue cells.

The term “specific cell lineage” as used herein is taken to refer to theancestry of a particular cell type, including ancestral cells and all ofthe subsequent cell divisions which occurred to produce the specificcell type. Differentiated stem cells of a specific cell lineage are agroup of cells that have the same cell type. Cells of the same cell typeare similar to each other, along with their associated intercellularsubstances, and perform the same function within a multicellularorganism. Cells of the same cell type preferably express a similar setof markers. Major tissue cell types include, but are not limited to,epithelial, endothelial connective, skeletal, muscular, glandular, andnervous tissues. In the present methods, the stem cells are preferablyco-cultured with tissue cells such that the stem cells are induced todifferentiate into a specific cell lineage that is the same cell type asthe tissue cells.

In the methods of the present invention a stem cell is undifferentiatedprior to culturing and is any cell capable of undergoingdifferentiation. The stem cell may be selected from the group including,but not limited to, embryonic stem cells, pluripotent stem cells,haematopoietic stem cells, totipotent stem cells, mesenchymal stemcells, neural stem cells, or adult stem cells. The stem cells arepreferably derived from a mammalian animal, most preferably, but notlimited to, a mouse or human.

The stem cells used in the methods of the present invention arepreferably embryonic stem (ES) cells. The stem cell is preferably amammalian embryonic stem cell which may be derived directly from anembryo, from a culture of embryonic stem cells, or from somatic nucleartransfer. Whilst, the stem cell may be derived from other mammaliananimals, they are most preferably human embryonic stem cells. Theembryonic stem (ES) cell used in the present method includes anembryonic cell derived from an embryo or a cell derived fromextraembryonic tissue. Suitable embryonic stem cells include those thatare commercially available such as those previously described (Reubinoffet al., 2000) or hES1, hES3, hES4, hES5, or hES6. These cells may beobtained from ES Cell International Pte Ltd.

The term “embryo” as used herein is defined as any stage afterfertilisation which can be up to 2 weeks post conception in mammals. Theembryonic period for mammals, such as a mouse is approximately 4-6 days.An embryo develops from repeated division of cells and includes thestages of a blastocyst stage which comprises an outer trophectoderm andan inner cell mass (ICM). The embryo may be an in vitro fertilisedembryo or it may be an embryo derived by transfer of a somatic cell orcell nucleus into an enucleated oocyte preferably of human or non-humanorigin. Extraembryonic tissue includes cells produced by the embryo thatmake up the placenta.

Suitable embryonic stem (ES) cells that may be used in the methods ofthe present invention may include mammalian ES cells. ES cells are knownto have pluripotent properties and may be induced to undergo controlleddifferentiation to produce diverse cell lineages in vitro.

The stem cells may be cultured in the presence of tissue cells to inducedifferentiation of the stem cells into a specific cell lineage. Theembryonic stem cells may be cultured in either methyl cellulosecontaining media in bacterial grade petri dishes or hanging drops toprevent their adherence to the surface of the culture dish, thusinducing the generation of colonies of differentiated cells known asembryoid bodies (EBs). EBs contain cellular representatives of all threeembryonic germ layers (ectoderm, mesoderm and endoderm) and underspecific culture conditions may be instructed and manipulated togenerate pure preparations of specific cell lineages.

The stem cells used in the present methods may be derived from anembryonic cell line, embryonic tissue, or somatic nuclear transfer. Theembryonic stem cells may be cells which have been cultured andmaintained in an undifferentiated state. The ES cells used may be eitheras a single cell suspension if intended for culture with a single cellsuspension of tissue sample. Alternatively, the ES cells may be grown ashanging drops in the presence of LIF such that they may formundifferentiated aggregates if intended for culture wrapped in aprepared tissue sheet. These aggregates are known as undifferentiatedembryoid bodies.

The stem cells suitable for use in the present methods may be derivedfrom a patient's own tissue. This would enhance compatibility ofdifferentiated tissue grafts derived from the stem cells with thepatient. The stem cells may be genetically modified prior to use throughintroduction of genes that may control their state of differentiationprior to, during or after their exposure to the embryonic cell orextracellular medium from an embryonic cell. They may be geneticallymodified through introduction of vectors expressing a selectable markerunder the control of a stem cell specific promoter such as Oct-4.

The stem cells may be genetically modified at any stage with markers sothat the markers are carried through to any stage of cultivation. Themarkers may be used to purify the differentiated or undifferentiatedstem cell populations at any stage of cultivation. Transgenic markers,for example, green fluorescent protein (GFP) allows for isolation ofpure stem cell derivatives utilising fluorescence activated sorting(FACs) at required lengths of time following induction. Differentiatedstem cells produced by the methods of the present invention may begenetically modified to bear mutations. Genetically modified stem cellsthat are induced to differentiate to specific cell lineages may beuseful culture models and may provide a route for delivery of genetherapy.

In the methods of the present invention the stem cell can be induced todifferentiate into a specific cell lineage, preferably selected from thegroup consisting of respiratory, pancreatic, mammary, renal, intestinal,hepatic, haematopoietic, muscle or cardiac cell lineages. Preferably,the stem cell is induced to differentiate into a respiratory celllineage.

The term “tissue sample” as used herein is taken to include, but not belimited to, tissue extracts, cell culture medium, biopsy specimens orresected tissue. The tissue sample, preferably includes tissue cells. Atissue sample, preferably includes tissue cells, that are a group ofcells similar to each other, along with their associated intercellularsubstances, which perform the same function within a multicellularorganism. Major tissue cell types include, but are not limited to,epithelial, endothelial connective, skeletal, muscular, glandular, andnervous tissues.

The tissue sample is preferably derived from a mammalian organism, mostpreferably a human subject. More preferably, the tissue sample is, butnot limited to, tissue derived from various mammalian organs, such as,respiratory, reproductive, kidney, brain, heart, muscle and skeletal.The tissue sample preferably includes tissue cells that are derived fromembryonic, foetal or post-partum tissue. It is preferred that a tissuesample having powerful inductive properties, such as foetal orpost-partum organs are used.

Most preferably, the tissue cells are mesenchyme cells. Mesenchyme cellsare derived from mesenchymal tissue, which is an embryonic connectivetissue, composed of cells contained within an extracellular matrix.Mesenchyme tissue harbors potent inductive signals that act to inducemore permissive cell populations to differentiate in a tissue specificmanner.

It has been unclear, however, whether any mesenchyme can instructdifferentiation of a primative cell line such as an embryonic stem cell,neural stem cell or mesenchymal stem cell line.

Without being limited by theory, it is likely that adult epithelialcontain a pluirpotent population of epithelial cells, which mightrepresent the adult stem cell population that has the capacity to giverise to an entirely new organotypic phenotype in response to theinductive and instructive signals from the mesenchyme. However, the ideathat the inductive and instructive properties of the mesenchyme aresufficient to direct differentiation of embryonic stem cells waspreviously unknown and was unexpected.

The tissue cells suitable for use in the methods of the presentinvention as applied to differentiation of lung are preferably derivedfrom lung mesenchyme, embryonic tissue. The tissue cells may preferablybe whole lung tissue sample, lung epithelium and/or mesenchyme assheets, or lung mesenchyme and/or epithelium in single cell suspensions.In the methods of the present invention, the culturing step may includeembedment techniques involving foetal or post-partum tissue samples(either whole tissue sources or parts of tissue, including epithelial ormesenchymal tissues).

It is most preferred that that tissues are selected duringorganogenesis, preferably when the organ of interest is actuallydeveloping. Once the period is identified for the organ, an optimaldevelopment period may be determined to select tissue fordifferentiation of the ES cells. Such optimisation may be conducted byknowing the tissue type and developmental periods and selecting tissuesfrom partitioned time periods. Pseudoglandular and canalicular stage aremost preferred as optimal stages for instructing respiratory lineagedifferentiation in stem cells.

Certain tissue types display organogenesis and undergo this process.Accordingly, it is within the scope of this invention that any tissuethat undergoes organogenesis and can be cultured to induce organogenesisin the presence of the stem cells can be used to induce differentiationof the stem cells to a specific cell lineage. It is most preferred thatthe tissues are selected from the group including, but not limited to,respiratory, pancreatic, mammary, renal, intestinal or hepatic tissues.

The term “extracellular medium” as used herein is taken to meanconditioned medium produced from growing a tissue cell as hereinbeforedescribed in a medium for a period of time so that extracellularfactors, such as secreted proteins, produced by the tissue cell arepresent in the conditioned medium. The medium can include componentsthat encourage the growth of the cells, for example basal medium such asDulbecco's minimum essential medium, BGJB—Fitton Jackson modifiedmedium, Ham's F12, or foetal calf serum. The extracellular medium maypreferably include cellular factors, such as secreted proteins, that arecapable of inducing differentiation of a stem cell. Such secretedproteins will typically bind receptors on a cell surface to triggerintracellular pathways which can initiate differentiation of the cell.Examples of suitable extracellular factors include HGF and FGF. Theextracellular medium may also contain polar molecules such as steroidswhich may pass through the cellular and/or nuclear membrane andassociate with intracellular factors which trigger a response andinitiate differentiation of the cell. Examples of suitable polarmolecules include retinoids, glucocorticoids.

The tissue cells and/or the stem cells used in the methods of thepresent invention may be tagged. Preferably, the stem cells and/ortissue cells used express a transgenic marker protein that allows foridentification of differentiated stem cells. Double staining for areporter gene expressed by stem cells and tissue specific markers may beused to determine the portion of differentiated stem cells relative tothe inductive tissue cells in culture. For example, epithelial specificmarkers such as cytokeratins, mesenchymal markers such as vimentin orlineage specific markers such as surfactant protein C may be used.

In the methods of the present invention the culturing step may involveintroducing stem cells to a tissue cell monolayer produced byproliferation of the tissue cells in culture. Preferably, the tissuecell monolayer is grown to confluence and the stem cell is allowed togrow in the presence of extracellular medium of the tissue cells for aperiod of time sufficient to induce differentiation of the stem cell toa specific cell lineage, wherein the differentiated stem cell is thesame cell type as the tissue cells. Alternatively the stem cell isallowed to grow for a period of time sufficient to inducedifferentiation to an intermediate precursor state in respect to thefully differentiated tissue cell. Alternatively, the stem cell may beallowed to grow in culture containing the extracellular medium of thetissue cell(s), but not in the presence of the tissue cells(s). Thetissue cells and stem cells could be separated from each other by afilter or an acellular matrix such as agar.

Suitable conditions for inducing differentiated stem cells are thosewhich are preferably non-permissive for stem cell renewal, but do notkill stem cells or drive them to differentiate exclusively intoextraembryonic cell lineages. A gradual withdrawal from optimalconditions for stem cell growth favours differentiation of the stem cellto specific cell types.

Suitable culture conditions may include the addition of retinoids,glucocorticoids, (as above) or growth factors in co-culture which couldincrease differentiation rate and/or efficiency. For instance, FIGS. 2and 3 demonstrate that such growth factors can induce murine ES andneural stem cells to undergo respiratory lineage differentiation invitro.

Other suitable culturing conditions would include consideration offactors such as cell density. If the tissue cells are plated, then it ispreferable that they are grown to confluence. The stem cells may then bepreferably dispersed and then introduced to a monolayer of tissue cells.The monolayer is preferably grown to confluence in a suitable medium,such as DMEM or M16 medium. More preferably, the stem cells and tissuecells are co-cultured until a substantial portion of the stem cells havedifferentiated.

In another aspect of the present invention there is provided a method ofinducing differentiation of a stem cell into a specific cell lineage,the method including the steps of:

-   -   mixing a first sample of stem cells with a second sample of        tissue cells, said cells selected during organogenesis of the        tissue to form a cell mixture;    -   culturing the cell mixture in vitro, under conditions that        induce differentiation of a stem cell into a specific cell        lineage.

Preferably, the tissue cells are in a substantially single cellsuspension prior to mixing with the stem cells. Alternatively, thetissue cells are prepared as a sheet for wrapping the undifferentiatedembryoid body. Preferably, the culturing step includes allowing the cellmixture to grow on a permeable membrane, wherein the membrane is incontact with a culture medium, such that the stem cells are induced todifferentiate into a specific cell lineage. It is preferred that thepermeable membrane be one that may float on the culture medium and thatthe cell mixture be placed at the air interface. Membranes suitable forsuch a purpose are millipore or nucleopore filters that preferably havea pore size of less than 0.22 μm.

In another aspect of the present invention there is provided a method ofproducing differentiated stem cells of a specific cell lineage, themethod including:

-   -   culturing stem cells in vitro in the presence of a tissue sample        and/or extracellular medium of a tissue sample, said sample        selected during organogenesis of the tissue, under conditions        that induce differentiation of a stem cell into a specific cell        lineage; and    -   recovering differentiated stem cells of a specific cell lineage.

Preferably, the tissue sample is treated to form tissue cells in asubstantially single cell suspension prior to culturing with the stemcells. Alternatively, the tissue cells are prepared as a sheet forwrapping an undifferentiated embryoid body.

Pure differentiated stem cells may be recovered by FACS if either thestem cell or the inducing tissue contains a fluorescent marker such asGFP. Alternatively, if the inducing tissue is grown on the opposingsurface of a filter to the stem cells, then pure populations ofdifferentiated stem cells may be recovered by mechanical disassociationfrom the filter.

In a preferred aspect of the present invention there is provided amethod of producing differentiated stem cells of a specific celllineage, the method including:

-   -   culturing stem cells in vitro in the presence of tissue cells,        said cells selected during organogenesis of the tissue, under        conditions that induce differentiation of the stem cell into a        specific cell lineage; and    -   recovering differentiated stem cells of a specific cell lineage.

Preferably, the tissue cells are in a substantially single cellsuspension prior to culturing with the stem cells. Alternatively, thetissue cells are prepared as a sheet for wrapping an undifferentiatedembryoid body.

The culturing step preferably includes allowing the stem cells to growon a first surface of a permeable membrane and allowing the tissue cellsto grow on an opposite second surface of the permeable membrane, whereinthe membrane is in contact with a culture medium, such that the stemcells are induced to differentiate into a specific cell lineage.Differentiated stem cells of a specific cell lineage may then berecovered from the first surface of the permeable membrane. Thepermeable membrane is preferably, but not limited to a transfiltermembrane, where inducing tissue cells and stem cells are placed onopposing sides of the membrane filter.

In the methods of the present invention the stem cells and tissue cellsneed not be in direct cell-cell contact with one another in culture. Thestem cells and tissue cells may be separated by a permeable membranethat allows the diffusion of soluble transmissible signals across themembrane. Suitable permeable membranes may preferably includetransfilter membrane, such as millipore or nucleopore filters. The useof a transfilter membrane in the cultures as hereinbefore describedprovides a convenient and efficient means for obtaining separated andpure populations of induced differentiated stem cells of a specific celllineage.

In order to facilitate the isolation of pure differentiated stem cellsof a specific lineage, heterotypic recombinations of differentiated stemcells and inductive tissue cells as hereinbefore described may beseparated by a permeable membrane, such as a nucleopore or milliporefilter. Double staining may also be performed to assess the specificcell type of the differentiated stem cell.

In the methods as hereinbefore described, the tissue cells may bederived from embryonic, foetal or post-partum tissue. Preferably, thetissue cells are embryonic mesenchymal cells. More preferably, thetissue cells are derived from lung mesenchyme tissue. The stem cellsused in the methods of the present invention are preferably embryonicstem (ES) cells. The tissue cells and/or the stem cells used in themethods of the present invention may be tagged. Preferably, the stemcells used express a transgenic marker protein that allows foridentification of differentiated stem cells. The stem cells may beinduced to differentiate into specific cell lineages, preferablyselected from the group consisting of respiratory, pancreatic, mammary,renal, intestinal, and hepatic.

In the methods of the present invention, the culturing step maypreferably include the addition of a growth factor to enhance stem celldifferentiation. Suitable growth factors may be preferably selected fromepidermal growth factor (EGF), hepatocyte growth factor (HGF) andfibroblast growth factors (FGFs) or steroid hormones (for example,glucocorticoids, vitamin A, thyroid hormone, and retinoids), or othersuitable growth enhancing factors such as insulin, serum and choleratoxin. For example, to enhance differentiation of stem cells into celllineages, growth factors such as FGF may be added to the culture.

In yet another aspect of the invention, there is provided differentiatedstem cell of a specific cell lineage produced according to the methodsas hereinbefore described. Preferably, the differentiated stem cell is,but not limited to, a lung, kidney, pancreatic, mammary, cardiomyocyte,skeletal muscle cell, , intestinal cell, liver cell, or a haematopoieticcell. The present invention also provides differentiated stem cellsproduced according to the methods of the invention that may be used fortissue repair, transplantation, cell therapy or gene therapy.

The methods of the present invention also provide a basis for developingcell-based treatments for tissue specific disorders, such as respiratoryspecific disorders including cystic fibrosis, emphysema, chronicbronchitis, congenital lung hypoplasias and viral infections. Forexample, stem cells may be co-cultured with lung tissue cells to obtainstem cells differentiated into an intermediate respiratory cell lineage.Intermediate cell lineages would represent any cell type in a stagebetween derivation from the embryonic inner cell mass, and prior toterminal differentiation of the desired cell type. The intermediatelydifferentiated stem cells may then be propagated to expand numbers.Intermediate cells may be then terminally differentiated in a culturedish for drug discovery programs. Alternatively, the intermediatelydifferentiated stem cells may be transferred to a host (i.e. forexample, mouse or human afflicted with a respiratory disease) in acellular replacement therapy requiring replacement of damaged orsub-optimally functioning respiratory tissue in vivo.

The differentiated cells and their intermediates may be used as a sourcefor isolation or identification of novel gene products including but notlimited to growth factors, differentiation factors or factorscontrolling tissue regeneration, or they may be used for the generationof antibodies against novel epitopes.

The differentiated cells produced according to the methods of thepresent invention may be clonally expanded. A specific differentiatedcell type can be selectively cultivated from a mixture of other celltypes and subsequently propagated. Specific differentiated cell typesthat are clonally expanded can be useful for various applications suchas the production of sufficient cells for transplantation therapy, forthe production of sufficient RNA for gene discovery studies etc. Thedifferentiated cells may be used to establish cell lines according toconventional methods.

The differentiated cells produced according to the methods of thepresent invention may be genetically modified. For instance, a geneticconstruct may be inserted to a differentiated cell at any stage ofcultivation. The genetically modified cell may be used aftertransplantation to carry and express genes in target organs in thecourse of gene therapy.

The differentiated stem cells produced according to the methods of thepresent invention may be preserved or maintained by any methods suitablefor storage of biological material. Effective preservation ofdifferentiated cells is highly important as it allows for continuedstorage of the cells for multiple future usage. Traditional slowfreezing methods, commonly utilised for the cryo-preservation of celllines, may be used to cryo-preserve differentiated cells.

The present invention further provides a cell composition including adifferentiated cell produced by the method of the present invention, anda carrier. The carrier may be any physiologically acceptable carrierthat maintains the cells. It may be PBS or other minimum essentialmedium known to those skilled in the field. The cell composition of thepresent invention can be used for biological analysis or medicalpurposes, such as transplantation. In addition, the cell composition ofthe present invention can be used in methods of treating diseases orconditions, such as respiratory disease.

The present invention will now be more fully described with reference tothe accompanying examples and drawings. It should be understood, howeverthat the description following is illustrative only and should not betaken in any way as a restriction on the generality of the inventiondescribed above.

EXAMPLES Example 1 Differentiation of Embryonic Stem Cells into LungAggregates

Between 4 and 8 foetal mouse lungs were collected on either embryonicday 11.5 (E11.5), E12.5 or E13.5 in PBS and transferred to 0.25%trypsin/ 0.04% EDTA for mechanical dissociation into single cellsuspension by aspiration using a p200 Gilson pipette and pipette tip.Trypsin was deactivated by washing in DMEM (GIBCO) containing 10% foetalcalf serum (FCS). Cells and medium were transferred to a 1.5 mleppendorf tube for centrifugation at 300 g for four minutes. Thesupernatant was decanted and the cell pellet resuspended in 300 μl ofBGJB Fitton Jackson modified media (GIBCO) containing 150 μg/ml ascorbicacid and supplemented with 5% FCS and 2 mM L-glutamine (hereafterreferred to as culture media).

Between 20,000 or 10,000 ZIN40 or green fluorescent protein (GFP)embryonic stem (ES) cells were then mixed gently with the 300 μlembryonic lung cell suspension. The cell mixture was then centrifugedagain for 4 minutes at 300 g and resuspended in 1 μl of culture media.This 1 μl of concentrated cell suspension was then transferred onto amillipore filter floating on pre-equilibrated culture media using afinely drawn glass pipette and incubated at 37° C. in 5% CO2. Culturemedia was changed every 2 days. After the designated culture period,embryonic lung/ES cell aggregates were peeled from the millipore filterswith forceps and transferred to a 2 ml round bottom eppendorf tubecontaining phosphate buffered saline (PBS). Aggregates were washed inPBS 3 times to remove culture media and fixed for 1 hour at 4° C. in 4%paraformaldehyde on a rocking stage. Aggregates were then once againwashed in PBS 3 times to remove fixative and processed for cellularanalysis.

Example 2 Differentiation of Neural Stem Cells into a RespiratoryLineage

Neurospheres derived in culture from green fluorescent protein (GFP)transgenic foetal mouse brains were analysed for the expression of theneural stem (NS) cell markers nestin and musashi. Zin 40 ES cells andEBs were cultured as previously described (Munsie et al., 2000). Embryoswere recovered from E12.5 EGFP +/− mouse matings (Jackson laboratories)and EGFP positive neurospheres generated according to Reynolds and Weiss(1992).

After four days of culture, either 50 embryoid bodies or 50 neurosphereswere collected and placed in a 35 mm culture dish in DMEM. Either HGF orNGF was added at a final concentration of 20 ng/ml and 100 ng/ml to thestem cells for eight days following plating in 35 mm dishes. In analternative and separate treatment, the embryoid bodies or neurosphereswere treated with DMEM supplemented with HGF+ media (DMEM containing 3%charcoal stripped foetal calf serum, 10 μg/ml insulin, 1 μg/ml choleratoxin, 25 ng/ml epidermal growth factor, 10 ng/ml hepatocyte growthfactor and 25 ng/ml FGF7). Cells were stained according to standardprocedures.

Results

Aggregates of foetal mouse lung induced immunoreactivity to therespiratory-specific marker SPC of both mouse and human ES cells. Of allmouse ES cell derivatives cultured as aggregates, 28% demonstratedimmunoreactivity to the SPC-specific antibody. Although only few humanES cell derivatives cultured as aggregates demonstrated immunoreactivityto the SPC-specific antibody, culture conditions remain to be optimisedand exact numbers undergoing respiratory-specific differentiation remainto be determined. Both mouse and human SPC immunoreactive ES cellderivatives in aggregates incorporated as tubule structures reminiscentof functional respiratory tubules. Tubules were composed either solelyof ES cell derivatives or of a mixture of contaminating endogenousrespiratory epithelia and ES cell derivatives. Furthermore, ES cellderivative SPC localisation was found to be polarised to the apicalsurface, indicative of normal functional respiratory cell types.

Both mouse and human ES cells cultured alone as embryoid bodiesdifferentiated into derivatives of three germ layers (mesoderm, ectodermand endoderm; data not shown), thus evidencing their ability to form alltissues of the body. However, we are as yet to identify SPCimmunoreactive ES cell derivatives in these aggregates at anysignificant level. Because our aggregate system results in the inductionof mouse ES cell derivative SPC immunoreactivity at an incidence of 28%,our aggregate system can thus be said to direct differentiation of bothES cells.

The morphology of EBs and neurospheres plated at high density in eitherHGF or NGF was similar to that of EBs and neurospheres cultured incontrol media after 8 days (data not shown). Neurospheres cultured inHGF+ media demonstrated a relatively reduced propensity to plate down asa monolayer when compared to controls, instead forming de novo fociwithin the dish (FIGS. 3A,B). Double Hoechst staining (nuclear) andanti-surfactant C immunocytochemistry (cytoplasmic) demonstrated thatmany of these foci contained surfactant C Ab reactive cells (FIGS.3C,D). Following culture at high density, RT-PCR demonstratedamplification of α-fetoprotein (an endoderm-specific marker) and Nkx2.1(an early marker of lung, thyroid, pituitary and diencephelondevelopment) transcripts in all culture conditions (FIG. 4).Semi-quantitative PCR demonstrated upregulation of Surfactant Atranscription within neurospheres cultured in the presence of either NGFor HGF+ media. Similarly, upregulation of Surfactant C transcription wasobserved in both EBs and neurospheres cultured in the presence of HGF+medium, whereas Surfactant D transcription was upregulated by treatmentwith HGF, NGF or HGF+ medium. Although α1-antitrypsin and clara cellsecretory protein (CC10) transcripts were detected in all EB cultureconditions, they were not detected in neurospheres under the cultureconditions tested. These studies demonstrate that in vitro, NS and EScells can be induced to express marker representatives of fullydifferentiated respiratory lineages.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises”, is not intended to exclude other additives, components,integers or steps.

The discussion of prior art documents, acts, devices and the like isincluded in this specification solely for the purpose of providing acontext for the present invention. It is not suggested or representedthat any or all of these matters formed part of the prior art base orwere common general knowledge in the field relevant to the presentinvention as it existed in Australia before the filing date of thisapplication.

Finally, the invention as hereinbefore described is susceptible tovariations, modifications and/or additions other than those specificallydescribed and it is understood that the invention includes all suchvariations, modifications and/or additions which may be made it is to beunderstood that various other modifications and/or additions which fallwithin the scope of the description as hereinbefore described.

REFERENCES

Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. (2000) Embryonicstem cell lines from human blastocysts: somatic differentiation invitro. Nat Biotechnol. 18(4):399-404.

1-35. (canceled)
 36. A method of inducing differentiation of a stem cellinto a specific cell lineage, comprising: culturing a stem cell in vitrounder conditions that induce differentiation of the stem cell into aspecific cell lineage, said step of culturing taking place in thepresence of at least one of (i) a tissue sample from a tissue, and (ii)extracellular medium from a tissue sample from a tissue, wherein saidtissue sample is selected during organogenesis of the tissue.
 37. Amethod of treating a tissue specific disorder, comprising: (a) obtaininga differentiated stem cell or a product of a differentiated stem cellaccording to a method that comprises culturing a stem cell in vitrounder conditions that induce differentiation of the stem cell into aspecific cell lineage, said step of culturing taking place in thepresence of at least one of (i) a tissue sample from a tissue, and (ii)extracellular medium from a tissue sample from a tissue, wherein saidtissue sample is selected during organogenesis of the tissue, andwherein said differentiated stem cell corresponds to a tissue of atissue specific disorder; and (b) administering the differentiated stemcell to a corresponding tissue of the tissue specific disorder in apatient in need.
 38. The method of claim 37 wherein the differentiatedstem cell is selected from the group consisting of a lung cell, a kidneycell, a pancreatic cell, a mammary cell, a cardiomyocyte, an intestinalcell, a hepatic cell, a brain cell, a muscle cell and a reproductivecell.
 39. The method of claim 37 wherein the differentiated stem cell isfrom a lung cell lineage.
 40. The method of claim 37 wherein thedifferentiated stem cell is genetically modified.
 41. A method oftreating a tissue specific disorder, comprising: obtaining anintermediate differentiated stem cell or a product of an intermediatedifferentiated stem cell according to a method that comprises culturinga stem cell in vitro under conditions that induce differentiation of thestem cell into a specific cell lineage to produce said intermediatedifferentiated stem cell, said step of culturing taking place in thepresence of at least one of (i) a tissue sample from a tissue, and (ii)extracellular medium from a tissue sample from a tissue, wherein saidtissue sample is selected during organogenesis of the tissue, whereinsaid intermediate differentiated stem cell is partially differentiatedfrom an embryonic inner cell mass stage to a terminally differentiatedstage, and wherein said stem cell corresponds to a tissue of the tissuespecific disorder; and administering the differentiated stem cell to thecorresponding tissue of the tissue specific disorder in a patient inneed.
 42. The method of claim 41 wherein the intermediate differentiatedstem cell is selected from the group consisting of a lung cell, a kidneycell, a pancreatic cell, a mammary cell, a cardiomyocyte, an intestinalcell, a hepatic cell, a brain cell, a muscle cell and a reproductivecell.
 43. The method of claim 41 wherein the intermediate differentiatedstem cell is from a lung cell lineage.
 44. The method of claim 41wherein the intermediate differentiated stem cell is geneticallymodified.
 45. The method of any one of claims 36, 37 or 41 wherein thestem cell is selected from the group consisting of an embryonic stemcell, a pluripotent stem cell, a haematopoietic stem cell, a totipotentstem cell, a mesenchymal stem cell, a neural stem cell, an adult stemcell and a genetically modified stem cell, wherein the geneticallymodified stem cell is selected from the group consisting of an embryonicstem cell, a pluripotent stem cell, a haematopoietic stem cell, atotipotent stem cell, a mesenchymal stem cell, a neural stem cell and anadult stem cell.
 46. The method of any one of claims 36, 37 or 41wherein the stem cell is derived from an embryonic cell line or anembryonic tissue, or by somatic nuclear transfer.
 47. The method of anyone of claims 36, 37 or 41 wherein the stem cell comprises an embryonicstem cell.
 48. The method of claim 47 wherein the embryonic stem cellcomprises a human embryonic stem cell or a mouse embryonic stem cell.49. The method of any one of claims 36, 37 or 41 wherein the stem cellcomprises a human embryonic stem cell.
 50. The method of any one ofclaims 36, 37 or 41 wherein the tissue sample and the stem cell expresssimilar markers.
 51. The method of any one of claims 36, 37 or 41wherein the tissue sample comprises a single cell suspension.
 52. Themethod of any one of claims 36, 37 or 41 wherein the tissue sample isselected at a pseudoglandular or canalicular stage of development duringorganogenesis.
 53. The method of any one of claims 36, 37 or 41 whereinthe tissue sample comprises a tissue cell type selected from the groupconsisting of an epithelial tissue cell, an endothelial connectivetissue cell, a skeletal tissue cell, a muscle tissue cell, a glandulartissue cell, and a nervous tissue cell.
 54. The method of claim 53wherein the tissue cell type comprises a mesenchymal cell.
 55. Themethod of any one of claims 36, 37 or 41 wherein the tissue sample isderived from an organ selected from the group consisting of arespiratory organ, prostate gland, a reproductive organ, kidney,pancreas, mammary gland, brain, heart, muscle, liver, and intestine. 56.The method of any one of claims 36, 37 or 41 wherein the tissue samplecomprises a mixture of mesenchymal tissue and epithelial tissue.
 57. Themethod of claim 56 wherein the tissue sample comprises fetal mesenchymaltissue or a mixture of fetal mesenchymal and epithelial tissue
 58. Themethod of any one of claims 36, 37 or 41 wherein the specific celllineage is selected from the group consisting of respiratory celllineage, pancreatic cell lineage, mammary cell lineage, renal celllineage, intestinal cell lineage, hepatic cell lineage, neural celllineage, cardiac cell lineage, and muscle cell lineage.
 59. The methodof any one of claims 36, 37 or 41 wherein the specific cell lineage is arespiratory cell lineage.
 60. The method of any one of claims 36, 37 or41 wherein the stem cell and the tissue sample are co-cultured but arenot in direct cell-cell contact.
 61. The method according to claim 60wherein the stem cell and the tissue sample are separated by a permeablemembrane.
 62. The method according to claim 61 wherein the permeablemembrane is a millipore filter or a nucleopore filter.
 63. The method ofeither claim 37 or claim 41 wherein the tissue specific disorder isselected from the group consisting of cystic fibrosis, emphysema,chronic bronchitis, congenital lung hypoplasia and viral infection. 64.A differentiated stem cell of a specific stem cell lineage that isprepared by a method which comprises culturing a stem cell in vitrounder conditions that induce differentiation of the stem cell into aspecific cell lineage, said step of culturing taking place in thepresence of at least one of (i) a tissue sample from a tissue, and (ii)extracellular medium from a tissue sample from a tissue, wherein saidtissue sample is selected during organogenesis of the tissue.
 65. Adifferentiated stem cell according to claim 64 that is selected from thegroup consisting of a lung cell, a kidney cell, a pancreatic cell, amammary cell, a cardiomyocyte, an intestinal cell, a hepatic cell, abrain cell, a muscle cell and a reproductive cell.
 66. A differentiatedstem cell according to claim 64 that is from a lung cell lineage.
 67. Adifferentiated stem cell according to claim 64 that is geneticallymodified.
 68. A cell composition comprising a differentiated stem cellaccording to any one of claims 64-66.
 69. An intermediate differentiatedstem cell of a specific stem cell lineage prepared by a method whichcomprises culturing a stem cell in vitro under conditions that inducedifferentiation of the stem cell into a specific cell lineage to producesaid intermediate differentiated stem cell, said step of culturingtaking place in the presence of at least one of (i) a tissue sample froma tissue, and (ii) extracellular medium from a tissue sample from atissue, wherein said tissue sample is selected during organogenesis ofthe tissue, and wherein said intermediate differentiated stem cell ispartially differentiated from an embryonic inner cell mass stage to aterminally differentiated stage.
 70. An intermediate differentiated stemcell according to claim 69 that is selected from the group consisting ofa lung cell, a kidney cell, a pancreatic cell, a mammary cell, acardiomyocyte, an intestinal cell, a hepatic cell, a brain cell, amuscle cell and a reproductive cell.
 71. An intermediate differentiatedstem cell according to claim 69 which is from a lung cell lineage. 72.An intermediate differentiated stem cell according to claim 69 which isgenetically modified.
 73. A cell composition comprising an intermediatedifferentiated stem cell according to any one of claims 69-72.